The focus of our research is to determine how signal transduction regulates directed motility and behavior in the bacterium Myxococcus xanthus using an integrated approach that combines biochemistry, genetics, and cell biology. M. xanthus is an excellent model system to address fundamental questions concerning cell-cell signaling and directed movement as cells form multicellular biofilms and fruiting bodies as part of a complex life cycle. M. xanthus fruiting bodies are similar in many ways to biofilms formed by Pseudomonas aeruginosa and are of public health interest since biofilms render bacteria resistant to antibiotics and are very difficult to treat in patients. Biofilm and fruiting body formation require the activity of chemosensory systems to direct cell movements. Previously, we have shown that the Frz chemosensory pathway regulates both vegetative swarming and developmental aggregation by controlling the reversal frequency of cells. Cell reversals in Myxococcus, like tumbling in flagellated bacteria, allows cells to reorient themselves and to bias directional motility based on the temporal sensing of stimuli. In this proposal we plan to study the localization of the Frz pathway proteins and how localization may impact cell reversals. We also plan to study feedback regulation in the pathway and the link between the Frz chemosensory system and the two gliding motility engines. The following specific aims are proposed: (i) Localization and analysis of Frz receptors - FrzCD is a cytoplasmic protein localized in clusters (arrays) that continuously rearrange and move around the cells. We plan to identify FrzCD associated proteins and how they might affect cluster function and possible roles for cytoskeletal proteins in cluster localization and mobility. (ii) Feedback regulation in the Frz pathway - We plan to study the regulation of site-specific FrzCD methylation, especially during development. We will identify methylation sites by mass spectrometry and the role of the methyltransferase (FrzF) in controlling methylation. We also plan to study FrzE, a CheA (histidine kinase)-response regulator fusion protein another potential site for feedback regulation. (iii) Coupling the Frz system to motility engines - We plan to study the roles of AglZ, FrzS, and MglA as possible links between the Frz system and the A- and S-motility systems and to screen for mutants that may be defective in this link. PUBLIC HEALTH RELEVANCE: The focus of our research is to understand how signal transduction regulates motility and multicellular interactions in the bacterium Myxococcus xanthus. Myxococcus forms fruiting bodies that are similar in many ways to biofilms formed by Pseudomonas aeruginosa and other pathogens; these are of public health interest since biofilms render bacteria resistant to antibiotics and are very difficult to treat in patients. Biofilm formation usually requires motility, chemotaxis, type IV pili and extracellular polysaccharide matrix materials; these are more easily studied in Myxococcus, a non-pathogenic bacterium, but the results are directly relevant to the understanding and control of pathogenic bacteria with similar properties.